TSC patients develop multi-system disease including benign tumors of the brain, heart, skin and kidney. By age 10, 80% of children with TSC have renal angiomyolipomas and/or renal cysts. Mammalian target of rapamycin (mTOR) complex 1 (TORC1), which is activated in tuberous sclerosis complex (TSC), is a master regulator of cell growth, cellular metabolism, and autophagy. Treatment with TORC1 inhibitors partially decreases the size of TSC-associated brain and kidney lesions, but they regrow when treatment is stopped. Our central hypothesis is that dysregulation of autophagy and cellular metabolism plays a critical role in the pathogenesis of TSC and in the response of TSC-associated renal lesions to TORC1-targeted therapy. In Aims 1 and 2, we will test the hypothesis that low levels of autophagy in TSC2-deficient cells lead to a metabolic starvation phenotype, making TSC2-deficient cells hypersensitive to further autophagy inhibition. Consistent with this hypothesis, we have found that inhibiting autophagy induces metabolic dysregulation and decreases the in vivo growth of TSC2-deficient cells. In Aim 3, we will address the hypothesis that p62/sequestosome1-dependent signaling networks promote the growth and survival of TSC2-deficient cells. Consistent with this hypothesis, we have found that p62 accumulates in human angiomyolipomas and other TSC2-deficient cells as a consequence of low autophagy, and that shRNA down regulation of p62 inhibits the in vivo growth of TSC2-deficient cells. Our in vivo strategy (Aims 2 and 3) includes TSC2-deficient angiomyolipoma-derived cells and renal cyst/cystadenoma quantitation in Tsc2+/- mice. Throughout the proposal, we will utilize innovative, state-of-the-art technology including real-time monitoring of bioenergetic parameters using the Seahorse system, metabolomic profiling in collaboration with the Broad Institute, high throughput synthetic-lethality drug screening, and in vivo bioluminescent detection of angiomyolipoma cells. Novel reagents will be generated, including 2 new mouse models (Tsc2+/-Atg5+/- and Tsc2+/-p62-/-.) and angiomyolipoma-derived cells with shRNA down regulation of key molecules. The significance of this project is that it will reveal for the firsttime how autophagy-dependent cellular networks contribute to the pathogenesis of renal disease in TSC. We expect this project to have high impact because TSC-associated lesions have devastating consequences in both children and adults and because the TORC1 signaling network is dysregulated in other human diseases including polycystic kidney disease.